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Design of a highly thermotolerant, immunogenic SARS-CoV-2 spike fragment immunogen
Sameer Kumar Malladi1, Randhir Singh2, Suman Pandey2, Savitha Gayathri2, Kawkab Kanjo1, Shahbaz
Ahmed1, Mohammad Suhail Khan1, Parismita Kalita1, Nidhi Girish2, Aditya Upadhyaya 2, Poorvi
Reddy2, Ishika Pramanick1, Munmun Bhasin1, Shailendra Mani3, Sankar Bhattacharyya3, Jeswin
Joseph4, Karthika Thankamani4, V. Stalin Raj4, Somnath Dutta1, Ramandeep Singh3, Gautham Nadig2,
Raghavan Varadarajan*1,5
1Molecular Biophysics Unit (MBU), Indian Institute of Science, Bengaluru, India
2Mynvax Private Limited, ES12, Entrepreneurship centre, SID, Indian Institute of Science, Bengaluru,
India
3Translational Health Science and Technology Institute, NCR Biotech Science Cluster, 3rd Milestone,
Gurugram-Faridabad Expressway, Faridabad-121001
4Virology Scientific Research (VSR) Laboratory, School of Biology, Indian Institute of Science
Education and Research Thiruvananthapuram (IISER TVM), Kerala, India.
5Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bengaluru, India
* Corresponding author: Raghavan Varadarajan
Email: varadar@iisc.ac.in, PHONE: +91-80-22932612, FAX: +91-80-23600535
Running title: Thermotolerant SARS-CoV-2 immunogen
Keywords: glycosylation, microbial, Pichia, thermostable, ACE2
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Abstract:
Virtually all SARS-CoV-2 vaccines currently in clinical testing are stored in a refrigerated or frozen state
prior to use. This is a major impediment to deployment in resource-poor settings. Several use viral vectors
or mRNA. In contrast to protein subunit vaccines, there is limited manufacturing expertise for these novel,
nucleic acid based modalities, especially in the developing world. Neutralizing antibodies, the clearest
known correlate of protection against SARS-CoV-2, are primarily directed against the Receptor Binding
Domain (RBD) of the viral spike protein. We describe a monomeric, glycan engineered RBD protein
fragment that is expressed at a purified yield of 214mg/L in unoptimized, mammalian cell culture and in
contrast to a stabilized spike ectodomain, is tolerant of exposure to temperatures as high as 100°C when
lyophilized, upto 70°C in solution and stable for over four weeks at 37°C. In prime:boost guinea pig
immunizations, when formulated with the MF59 like adjuvant AddaVax™, the RBD derivative elicited
neutralizing antibodies with an endpoint geometric mean titer of ~415 against replicative virus, comparing
favourably with several vaccine formulations currently in the clinic. These features of high yield, extreme
thermotolerance and satisfactory immunogenicity suggest that such RBD subunit vaccine formulations
hold great promise to combat COVID-19.
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Introduction
SARS-CoV-2 is the etiological agent of the on-going COVID-19 pandemic (1, 2). As on 4th October, 2020
there are ~34.7 million infections and ~1.0 million deaths worldwide (3). The major surface protein of
SARS-CoV-2 is the spike glycoprotein. Like several other viral surface glycoproteins, it is a homotrimer,
with each protomer consisting of two subunits S1 and S2. The S1 subunit consists of an N-terminal domain
(NTD), linker and receptor binding domain (RBD), and two small subdomains SD1 and SD2 (4–6) (Figure
1A-1D). The RBD domain of the spike glycoprotein binds to the cell surface receptor ACE2, followed by
endocytosis or fusion mediated via the fusion peptide located on the S2 subunit (7). Most of the
neutralizing antibody responses are targeted to the RBD (8–14), though very recently, neutralizing
antibodies against the NTD have also been identified (15). It is thus unclear whether the full length spike
or the RBD is a better immunogen.
Over 150 vaccine candidates are under development globally (16). Some vaccine candidates that have
entered rapidly into clinical phase testing include mRNA vaccine candidates by Moderna (mRNA-1273),
BioNTech (BNT162b1)(17), and CureVac (CVnCoV), a Chimpanzee Adenovirus vector vaccine by
University of Oxford and AstraZeneca (ChAdOx1-S) (18), a non-replicating adenovirus type-5 (Ad5)
vaccine by Cansino (Ad5-nCoV) (19), a DNA vaccine by Inovio (INO-4800) (20), inactivated virus
vaccines by Sinovac (PiCoVacc) (21) and Bharat Biotech (COVAXIN), a native like trimeric subunit
spike protein vaccine by Clover Biopharmaceuticals /GSK/Dynavax (SCB-2019), and a full length
recombinant glycoprotein nanoparticle vaccine by Novavax (NVX-CoV2373) (16, 22). The majority of
the above employ full length spike or the corresponding ectodomain as the antigen. While there is some
encouraging pre-clinical and Phase 1 clinical data, there is no precedent for use of mRNA or viral vectors,
which are the farthest along in clinical development, in mass human vaccinations. In addition, with
inactivated or attenuated virus, there are obvious safety issues that need careful attention. There are few
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studies that compare the relative immunogenicity of multiple vaccine candidates expressed in multiple
platforms (23). Herein, we report a mammalian cell expressed, glycan engineered, RBD based subunit
vaccine candidate (mRBD) formulated with an MF59 equivalent adjuvant. In contrast to an equivalent
Pichia pastoris expressed RBD protein formulation, mRBD elicits titers of neutralizing antibodies in
guinea pigs well above the levels required for protection in non-human primate challenge studies. mRBD
expresses at eight-fold higher levels and is substantially more tolerant to thermal stresses than a stabilized
spike ectodomain without compromising immunogenicity, and can be stored for over four weeks at 37°C.
These data suggest that it is a promising candidate for further clinical development.
14 685 686 1273816319 541
A
B C
E
SARS-CoV-2 331 NITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDL 390
SARS-CoV-1 318 NITNLCPFGEVFNATKFPSVYAWERKKISNCVADYSVLYNSTFFSTFKCYGVSATKLNDL 377
SARS-CoV-2 391 CFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYN 450
SARS-CoV-1 378 CFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYN 437
SARS-CoV-2 451 YLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRV 510
SARS-CoV-1 438 YKYRYLRHGKLRPFERDISNVPFSPDGKPCTP–PALNCYWPLNDYGFYTTTGIGYQPYRV 496
SARS-CoV-2 511 VVLSFELLHAPATVCGPKKSTN 532
SARS-CoV-1 497 VVLSFELLNAPATVCGPKLSTD 518
* ** *
* * * * ** ** ** * ** ***
RBM
RBM
* ** * *
D
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Figure 1: S-protein domain organization, structure of Spike and receptor binding domain of SARS-CoV-2. A) Linear map of the S protein spike with the following domains: NTD, N- terminal domain; L, linker region; RBD, receptor-binding domain; SD, subdomain; UH, upstream helix; FP, fusion peptide; CR, connecting region; HR, heptad repeat; CH, central helix; BH, β-hairpin; TM, transmembrane region/domain; CT, cytoplasmic tail. B) Spike ectodomain trimer highlighting protomer with RBD in the ‘up’ conformation, NTD in dark blue, RBD in brick red, SD1 and SD2 in green and S2 subunit in magenta (PDB: 6VSB) C) Epitopes for known RBD directed neutralizing antibodies. The N and C termini of the receptor- binding motif (RBM) are labeled and in green. Residues at the binding interfaces with hACE2 are in cyan. The B38 epitope has considerable overlap with the hAce2 interface, non-overlapping residues are in light blue. Epitopes for S309, P2B-2F6 are in orange and yellow. Epitope for CR3022 is in pink, this overlaps substantially with the potent neutralizing antibody H014. The conserved N-glycosylation site at 343 and the engineered, immune masking glycosylation site at 532 are shown in red. D) Exposed residues with solvent accessible surface area that are not part of any neutralizing epitope identified so far are shown in dark blue. The largest such stretch is at the C-terminus where the engineered glycosylation site is placed. E) Sequence alignment of SARS-CoV-1 (residues: 318-518) and SARS-CoV- 2 (residues 331-532), the blue line indicates the receptor binding motif (RBM), the grey highlight indicate residues conserved in both SARS-CoV-1 and SARS-CoV-2 and the blue asterisks indicate the ACE2 binding residues. (PDB: 6M0J)
Results
Design of a recombinant RBD subunit vaccine
The RBD of the spike protein is the major target of neutralizing antibodies (8, 10–14, 24). SARS-CoV-2
is 79.6% identical to SARS-CoV-1 sequences (25). The spike protein of SARS-CoV-2 is 80% identical to
its homolog from SARS-CoV-1. The RBD of SARS-CoV-2 shares 74% amino acid sequence identity
with the RBD of SARS-CoV-1. We hypothesized that a receptor binding domain subunit derivative that
lacks flexible termini as well as unpaired cysteines, and retains the ACE2 receptor binding site, located
within the receptor binding motif (RBM, comprising residues 438-505, Figure 1E) as well as the cryptic
epitope recognized by the neutralizing antibody CR3022 would be a good immunogen. We selected the
RBD residues based on SWISS Model structure-based modelling of SARS-CoV-2 sequence, prior to
availability of any SARS-CoV-2 spike and RBD-ACE2 complex structures. The modelled structure
closely resembles the recently determined experimental structures determined by X-ray crystallography
or Cryo-EM (RMSD: 1.2Å, with X-ray structure PDB: 6M0J) (5, 6, 26). In the X-ray structure, residues
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after 526 are disordered. Two RBD sequences were shortlisted consisting of residues 331-532 and 332-
532 with retention (m331RBD) or deletion (mRBD/pRBD) of the native glycan at N331 for expression in
mammalian and P. pastoris expression systems respectively. The constructs for mammalian expression
are designated as m331RBD and mRBD, and for Pichia expression, pRBD respectively. In the past few
months, several potent neutralizing antibodies directed against the RBD have been isolated and it currently
appears that virtually the entire exposed surface of the RBD is targeted by neutralizing antibodies, with
the exception of the C-terminal region distal from the RBM. We have introduced a glycosylation site at
N532 in all the above RBD constructs to mask this region of the surface (Figure 1C, 1D).
RBD (332-532) is more highly expressed and thermotolerant than a stabilized spike ectodomain
Mammalian cell expressed m331RBD and mRBD were purified by single step Ni-metal affinity
chromatography from transiently transfected Expi293F culture supernatants. The proteins were confirmed
to be predominantly monomeric by SEC (Figure 2A). Proteins from both the constructs were pure and
were expressed at yields of ~68 ± 10 mg/L and ~214 ± 9 mg/L for m331RBD and mRBD respectively.
Removal of the N-terminal glycan in m331RBD by introducing the T333H mutation resulted in
substantially increased expression, similar to that of mRBD, confirming that the presence of the N-
terminal glycan is responsible for reduced yield, as has been observed previously for SARS-CoV-1 RBD
(27). All proteins were monomeric. In SEC, m331RBD which has an additional glycan, elutes before
mRBD (Figure 2A). Given the higher yield of mRBD, most subsequent studies were carried out with this
RBD derivative. nanoDSF thermal melt studies demonstrated that removal of the N-terminal glycan did
not affect protein stability (Figure 2B). mRBD bound ACE2-hFc with a KD of about ~14.2 nM (Figure
2C) and the neutralizing antibody CR3022 with a KD of 16 nM, confirming that the molecule is properly
folded (Figure 2D). mRBD is digested by trypsin with approximate half-lives of 20 and 60 minutes at 37
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and 4 °C (Figure 2E) respectively. The digestion kinetics is unaffected by storage for over a week at 4
°C.
Figure 2: Characterization of mammalian cell expressed RBD A) Size exclusion chromatography
profile of m331RBD, mRBD immunogens with predominantly monomeric peak at ~16.0 and ~16.3mL
kon: 6.9x105 M-1s-1
koff: 1.1x10-2 s-1
KD: 15.9x10-9 M
-200 0 200 400 600 800
CR3022
A
C
B
kon: 7.1x105 M-1s-1
koff: 1.0x10-2 s-1
KD: 14.2x10-9 M
200
0
-200 0 200 600 800
100
Res
po
nse
Un
its
(RU
)
Time (s)
ACE2-hFc
D
0 2 5 10 20 30 60 L 0 2 5 10 20 30 60
37 C 4 C
250150100
75
50
37
25
20
15
10
kDadtt + + + + + + + + + + + + + +
E
Temperature (oC)
0 20 40 60 80 100
Fir
st D
eriv
ati
ve
(35
0n
m/3
30
nm
)
0.000
0.005
0.010
0.015
0.020
0.025
0.030m331RBD (Tm: 50.8 ± 1.5 °C)mRBD (Tm : 50.3 ± 0.8 °C)
Elution volume (ml)
0 5 10 15 20 25
UV
28
0 A
bso
rba
nce
(m
AU
)
0
20
40
60
80
100
120
140m331RBD
mRBD
0
100
200
300
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respectively on S200 10/300GL column calibrated with Biorad gel filtration marker (Cat.No. 1511901) run
at flowrate of 0.5mL/min with PBS (pH 7.4) as mobile phase B) nanoDSF equilibrium thermal unfolding
of m331RBD and mRBD. C) SPR binding sensorgrams to ACE2 receptor. The concentrations of mRBD
used as analytes are 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM D) SPR binding sensorgrams of mRBD
with the neutralizing antibody CR3022. mRBD analyte concentrations are 50 nM, 25 nM, 12.5 nM, 6.2
nM, and 3.1 nM. E) Limited proteolysis of purified mRBD protein by TPCK treated trypsin (RBD:TPCK
Trypsin=50:1) at 4°C and 37°C.
A construct with identical amino acid sequence to mRBD (pRBD) was expressed and purified from P.
pastoris strain X-33 from a stably integrated gene cassette at a yield of ~50 mg/L in shake flasks. The
Pichia protein is more heterogeneous, extensively glycosylated and elutes at higher molecular weight than
mRBD in both SDS-PAGE and SEC (Supplementary Figure 1A, 1F). The thermal stability of the Pichia
purified immunogen pRBD (Tm: 49.2 °C) is similar to mammalian cell expressed versions (Supplementary
Figure 1B). The protein bound with comparable affinity to ACE2-hFc and CR3022 with KD’s of
approximately 23 nM and 30 nM respectively, similar but slightly higher than corresponding values for
mRBD (Supplementary Figure 1D, 1E). Pichia expressed RBD was similarly stable to thermal stress and
proteolysis (Supplementary Figure 1C, 1F, 1G). We also attempted to express the protein in E. coli. The
protein expressed well but was targeted to inclusion bodies. Despite multiple attempts employing a variety
of refolding strategies, we were unable to obtain significant quantities of properly refolded protein,
competent to bind ACE2 from E. coli.
The spike ectodomain and full length spike formulations are important SARS-CoV-2 vaccine candidates
(5, 6, 22) and it is therefore important to compare mRBD with these. We purified the Spike-2P stabilized
ectodomain (Spike containing mutations K968P and V969P) protein from Expi293F cells by single step
nickel chelate affinity chromatography followed by tag removal with a purified yield of ~25 mg/L culture
(5). The purified protein was observed to be trimeric on SEC and bound tightly to ACE2-hFc with little
dissociation (Figure 3A, 3B). Negative-stain EM confirmed that Spike-2P purified by us adopts a native
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like elongated trimeric structure (Figure 3C) consistent with available structures determined by Cryo-EM
(5, 6, 15). Spike-2P was rapidly digested by trypsin with approximate half-lives of 10 and 30 minutes at
37 and 4 °C respectively (Figure 3D-E) yielding multiple RBD containing fragments.
Figure 3: Characterization of mammalian cell expressed Spike-2P A) Size exclusion chromatography
profile of Spike- 2P ectodomain with a trimeric peak at ~8.9 mL on S200 10/300GL column calibrated with
Biorad gel filtration marker (Cat. No. 1511901) run at flowrate of 0.75 mL/min with PBS (pH 7.4) as mobile
phase. B) SPR binding sensorgrams of Expi293F purified Spike-2P with immobilized ACE2-hFc. The
Elution volume (ml)
0 5 10 15 20 25
UV
28
0 A
bso
rba
nce
(m
AU
)
0
20
40
60
80Spike-2P
kon : 2.4x104 M-1s-1
koff : ND*
KD : ND*
Res
po
nse
Un
its
(RU
)
-200 0 200 400 600 8000
200
400
Time (s)
A C
B
D
100 nm
S1
S2
S1S2
25015010075
50
37
25
20
kDa
4 C37 C
0 2 5 10 20 30 60 0 2 5 10 20 30 60
+ + + + + + + L + + + + + + +0 2 5 10 20 30 60 0 2 5 10 20 30 60
+ + + + + + + L + + + + + + +β-ME
4 C37 C
E
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concentrations of Spike-2P analyte used are 146 nM, 73 nM, 36.5 nM, 18 nM, 9 nM. C) Negative staining
EM images of Spike-2P protein. TEM images indicate that the sample is homogeneous and
monodisperse. Representative 2D reference free class averages of Spike-2P protein. Well-defined class
averages indicate that the Spike-2P sample has a stable and ordered structure and enlarged views of two
class averages show the S1 and S2 subunits of spike protein. D) SDS-PAGE Coomassie stained gel
following limited proteolysis of purified Spike-2P by TPCK treated trypsin (RBD:TPCK Trypsin=50:1) at
4°C and 37°C. E) Western blot following limited proteolysis of purified Spike-2P by TPCK treated trypsin
(RBD:TPCK Trypsin=50:1) at 4°C and 37°C, probed by α-mRBD guinea pig sera. The red arrow denotes
the expected position of the RBD.
Maintaining a proper cold chain during mass vaccination programs can be challenging in low and middle
income countries (28, 29). The aggregation state of mRBD and Spike-2P proteins was unchanged upon
storage at 4°C, freeze thaw and hour-long 37 °C incubation (Supplementary Figure 2A, 2B). mRBD and
Spike were also incubated at various temperatures both in PBS buffer for 60 minutes and for 90 minutes
in the lyophilized state. Protein conformational integrity was then assessed in ACE2 binding experiments
using SPR. In PBS, mRBD is 20 °C more stable to thermal stress compared to Spike-2P (Figure 4A, 4B).
Remarkably, lyophilized mRBD was stable to exposure of temperatures as high as 100 °C whereas Spike-
2P rapidly lost activity at temperatures above 50 °C both in solution and in the lyophilized state (Figure
4C, 4D). In solution, mRBD thermal unfolding was highly reversible in contrast to Spike-2P, as assessed
by repetitive equilibrium thermal unfolding (Figure 4 E, 4F). mRBD had identical thermal stability
profiles before and after lyophilization (Supplementary Figure 2C). mRBD was also resistant to longer
time (16 hour) thermal stress and showed only small changes in the thermal unfolding profile when
incubated for this time at temperatures up to 70 °C in the lyophilized state, and up to 37 °C in pH 7.0
buffer (Figure 4G, 4H, Supplementary Figures 2D, 2E). Even after storage at 37 °C for four weeks in the
lyophilized state, the thermal stability as well the Ace2 binding of the protein were unaffected (Figure 4G,
4H)
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4 9950 60 70Temperature ( C)
400
200
0-200 0 200 400 600 800
Spike-2P
160
120
80
40
0-200 0 200 400 600 800
mRBD
30 40 50 60 70Temperature ( C)
-200 0 200 400 600 800
200
100
0
300
Spike-2P
-200 0 200 400 600 800
0
40
80
160
mRBD
Res
po
nse
Un
its
(RU
)
Time (s)
Protein in 1X PBS
Lyophilized protein
A
C
B
D
FE
Temperature (oC)
30 40 50 60 70
Fir
st D
eriv
ati
ve
(35
0n
m/3
30
nm
)
0.000
0.005
0.010
0.015
0.020
0.025mRBD U1
mRBD U2
Temperature (oC)
30 40 50 60 70
Fir
st D
eriv
ati
ve
(35
0n
m/3
30
nm
)
-0.002
-0.001
0.000
0.001
0.002
0.003
0.004
0.005
0.006
Spike-2P U1
Spike-2P U2
0.00
0.01
0.02
0.03
30.0 40.0 50.0 60.0 70.0
Fir
st D
eriv
ati
ve
(35
0n
m/3
30n
m)
Temperature ( C)
4°C, Water, Freshly Dialysed
Lyophilized 37°C, 1 Week
Lyophilized 37°C, 2 Week
Lyophilized 37°C, 4 Week
-200 0 200 400 600 800
0
40
80
120
4 C, Water, Freshly Dialysed
Lyophilized 4 C, 2 Week
Lyophilized 4 C, 4 Week
Lyophilized 37 C, 2 Week
Lyophilized 37 C, 4 Week
RU
Time (s)
G H
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Figure 4: RBD and Spike protein functionality upon subjecting to thermal stress. SPR sensorgrams of ACE2 binding by A), B) protein in 1X PBS, subjected to thermal stress for 60 minutes C), D) Lyophilized protein subjected to thermal stress for 90 minutes. 100 nM of Spike-2P and mRBD were used as analytes. E), F) Equilibrium thermal unfolding measured using nanoDSF for Spike-2P and mRBD. The initial and repeat unfolding scans are in red (U1) and blue (U2) respectively. G) Equilibrium thermal unfolding measured using nanoDSF for lyophilized mRBD subjected to 37°C incubation for up to four weeks. H) SPR sensorgrams of ACE2 binding by lyophilized mRBD incubated at 4°C, 37°C for up to four weeks. 100nM of mRBD in 1XPBS was used as analyte.
AddaVax™ adjuvanted RBD elicits neutralizing antibodies in guinea pigs, functionally blocking the
receptor binding motif
Guinea pigs are a widely used, outbred animal model for respiratory infectious diseases and display
disease susceptibility and immune responses that are more similar to humans than the mouse model (30).
Guinea pigs have also been used to evaluate other COVID-19 vaccine candidates (20, 31, 32). Guinea
pigs were immunized with mammalian cell expressed mRBD protein adjuvanted with AddaVax™.
AddaVax is a squalene-based oil-in-water emulsion that is a mimetic of MF59. MF59 has an extensive
safety record and has been used in millions of people in the context of adjuvanted influenza vaccines (33).
Animals were primed at day 0 and boosted at day 21 with bleeds at day -1 (Pre-Bleed), day 14 and day
35.
The end point ELISA titers to self-antigen ranged from 1:6400 to 1:102400 after the second immunization
in individual animals (Figure 5A). To further confirm and extend these results, the study was repeated
with the inclusion of two additional groups immunized with Pichia expressed RBD and mammalian cell
expressed Spike-2P in addition to the mRBD (Figure 5A, 5B). Results with the mRBD were consistent in
both studies (Figure 5A). Pichia expressed RBD was as immunogenic as the mRBD in terms of self-titers
(Supplementary Figure 3A, 3B) but sera reacted poorly with mRBD and Spike-2P. Several studies have
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now shown that ACE2 competition titers and neutralizing antibody titers are highly correlated (22, 34).
Hence serum competition assays were carried out (Figure 5C). Endpoint neutralization titers with
replicative virus were measured using cytopathic effect (CPE) as a readout for infection (Figure 5D) and
found to range from 160-1280. Surprisingly, the pRBD sera were non-neutralizing and poorly cross-
reactive with mRBD and Spike-2P proteins (Figure 5A, 5C, 5D), presumably because of
hyperglycosylation of the Pichia expressed protein. In Spike-2P immunized animals titers were more
variable than in mRBD immunized animals though the difference in neutralization titers did not approach
statistical significance. A potential advantage of using the spike as an immunogen is that it contains
neutralization epitopes outside the RBD, including in the NTD (35). We therefore probed the Spike-2P
sera for NTD titers using a mammalian cell expressed NTD construct. However, all spike sera had NTD
endpoint ELISA titers less than 100. mRBD elicited serum neutralization titers that compared favorably
with those observed with several vaccine modalities in a variety of different organisms including guinea
pigs (Figure 5E) (18, 19, 21, 36–41)(42). A recent study compared titers elicited by an inactivated virus
vaccine formulation (BBIBP-CorV, Figure 5E) in mice, rats, guinea pigs and non-human and non-human
primates, the data show close consistency across all the different animal models (31).
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Figure 5: Comparative immunogenicity data. A-D) Guinea Pig serum titers obtained after two immunizations with AddaVax™ formulated immunogens. A), B) ELISA endpoint titer against mammalian cell expressed RBD and Spike ectodomain respectively. C) 50% Inhibitory titers of ACE2 receptor competing antibodies from animals immunized with mRBD, pRBD and Spike-2P respectively. Competition titers below 10 are uniformly assigned a value of 5. The dashed line represents the value 10. D) Endpoint neutralization titers in a cytopathic effect (CPE) assay against infectious SARS-CoV-2, Isolate USA-WA1/2020. The dashed line represents the value 10. (●)Sera from the first batch of animals immunized sera with mRBD, (○) second batch of animals immunized with mRBD, (▲) animals immunized with pRBD, (▼) immunized with mammalian expressed Spike-2P. E) Live virus neutralization titers for various vaccine candidates in mice, macaques and humans. mRNA-1273: mRNA vaccine
Figure 6
E
Neu
tra
liza
tion
Tit
er
1
10
100
1000
10000
100000Guinea Pig Mice Macaque Human
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expressing full length Spike-2P protein assayed by PRNT (36, 37, 40). BNT162b1: nucleoside modified mRNA vaccine expressing RBD subunit fused to T4 Fibritin derived Foldon trimerization domain assayed by PRNT(17). NVX- CoV2373: Full length Spike-2P adjuvanted protein vaccine assayed by CPE(22, 43). ChAdOx1 nCoV-19: Replication-deficient Chimpanzee Adenovirus vector expressing spike protein assayed by PRNT and Marburg VN(18, 38, 39). Ad5-ncov: Replication- defective Adenovirus type 5 vector expressing spike protein assayed by CPE(19). Ad26.COV2.S: Replication-defective Adenovirus type 26 vector expressing spike protein assayed by PRNT (41, 44). PiccoVacc: Chemically inactivated SARS-CoV-2 virus vaccine assayed by CPE (21). BBIBP: Chemically inactivated SARS-CoV-2 virus vaccine assayed by CPE (31, 42). INO-4800: DNA vaccine expressing full length Spike-2P protein assayed by CPE (20). Macaque data in INO-4800 is obtained by a SARS-CoV-2 pseudovirus neutralization assay (45).
Discussion
The majority of SARS-CoV-2 vaccine candidates currently in clinical testing use either full length spike
or the corresponding ectodomain as antigen, and most involve relatively new nucleic acid or viral vector
modalities that have not been tested in large scale immunizations. There is an obvious need for highly
expressed, stable, low-cost and efficacious subunit vaccine formulations and for side-by-side comparisons
of different candidates. In the present study we characterized the comparative yield, stability and
immunogenicity of mammalian cell and Pichia expressed RBD, as well as mammalian cell expressed
stabilized Spike-2P protein. All three candidates were successfully expressed, properly folded and
immunogenic. The data clearly indicate mammalian cell expressed, glycan engineered RBD to be the best
of the three immunogens, displaying reversible thermal unfolding and exceptionally high thermal
tolerance and stability to storage at 37 °C upto at least four weeks, a very important attribute for
deployment in low resource settings. The ability of the mRBD to elicit neutralizing antibodies was
comparable to that of the spike ectodomain as also seen in a recent rabbit immunogenicity study with a
different RBD derivative comprising residues 319-541(23). The RBD fragment could also be expressed
at high yield in the microbial host Pichia pastoris, and was properly folded, stable and immunogenic.
Interestingly, an alhydrogel adjuvanted formulation of a related SARS-CoV-1 RBD construct was recently
shown to be immunogenic and protect mice from SARS-CoV-1 challenge (46). Unfortunately, in the
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present study when pRBD was used as an immunogen, the elicited antibodies were poorly reactive with
either the mammalian cell expressed RBD or the corresponding Spike ectodomain. Further, they failed to
block binding of RBD to the ACE2 receptor, suggesting that further alterations to the Pichia expressed
sequence or adjuvant, use of an alternative Pichia strain, or optimization of growth/fermentation
conditions are required before it can be used as an effective immunogen. Recently, various RBD derived
subunit vaccine candidates have been tested for immunogenicity employing varying fragment lengths,
fusion adaptors (Fc, dimers), and adjuvants. No antibody dependent enhancement of infection,
immunopathologies, or Th2 bias have been observed with the SARS-CoV-2 RBD subunit derivatives
examined so far (46–49). Three independent studies used RBD-Fc fusions with one study using RBD
residues 331-527, another used RBD-Fc from Sino Biologicals (residues not mentioned) and a third used
a full-length S1-Fc fusion (residues 14-685) reporting viral neutralizing antibody titers of ~100-400, 1280
and NT50 derived from pseudoviral neutralizations of 378 respectively (47, 48, 50). One study employed
a week long intra-peritoneal immunization regime that is difficult to implement in large-scale human
vaccination programs (50). The other studies utilizing RBD-Fc and S1-Fc (47, 48), employed Freunds
adjuvant, again not used in human vaccinations. For the present mRBD formulation both the IC50 values
in the ACE2 competition assay and the viral neutralization titers were about 2% of the corresponding
ELISA endpoint titers, suggesting that a significant fraction of the elicited antibodies are neutralizing.
Oligomerization and nanoparticle display strategies have proven to induce appreciably higher neutralizing
antibody titers than corresponding monomers, this could be potentially be exploited with our mRBD
construct in future studies (49, 51, 52). However the effect of these modifications, as well as the exact
choice of chain termini which differ between the various RBD constructs, on thermotolerance remain to
be studied. Additionally, display on a heterologous scaffold will likely elicit significant antibody titers
against the scaffold as well as the displayed immunogen, which might pose regulatory challenges. An
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mRNA vaccine encoding a longer RBD fragment (319-541) elicited approximately comparable
neutralization titers in mice and macaques to those observed in the present study. In the same study, a
corresponding luciferase reporter mRNA formulation was shown to tolerate 37 C incubation for a week
with ~13% loss in activity (53). Multiple studies employing a variety of vaccine formulations and
modalities have now demonstrated that SARS-CoV-2 viral neutralization titers in small animals, including
mice and guinea pigs, are predictive of immunogenicity in macaques and humans (Figure 5E) (17, 18, 40,
46–49, 54, 19, 20, 22, 23, 36–39). Despite promising immunogenicity in several cases, all of the above
liquid vaccine formulations were either refrigerated or frozen prior to use. In contrast, the mRBD
described above can be stored lyophilized without refrigeration for at least four weeks, and is also tolerant
to transient high temperature exposure. In future studies the present formulation will be tested for its
ability to confer protection against challenge in an appropriate model, following which it can be advanced
to clinical development. It will also be valuable to examine if other RBD protein formulations with
different chain termini are similarly thermotolerant.
Materials and Methods
SARS-CoV-2 RBD, NTD, Spike ectodomain and antibody expression constructs
Two fragments of the SARS-CoV-2 Spike protein (S) (accession number YP_009724390.1) consisting of
the receptor binding domain (RBD) residues 331-532 with an N-terminal glycosylation site and 332-532
with deletion of the N-terminal glycan site deletion were chosen. Residue N532 was engineered to be
glycosylated by introducing an NGS motif at the C-termini of the RBD into both immunogen sequences.
The resulting sequences with an HRV-3C precision protease cleavage site linked to a 10xHistidine tag by
GS linker was codon optimized for human cell expression were expressed under control of the CMV
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promoter with a tPA signal sequence for efficient secretion. The clones were named m331RBD (331-532)
and mRBD (332-532). Identical RBD amino acid sequences to those described above, codon optimized
for Pichia pastoris expression were cloned into the AOX1 promoter containing vector pPICZalphaA,
containing a MATalpha signal sequence for efficient secretion. The resulting clones were named
p331RBD (expressing RBD 331-532) and pRBD (expressing RBD 332-532). In the present study, only
pRBD was utilized, based on expression data for the corresponding mammalian and insect cell expression
clones. A spike N-terminal domain construct (NTD) (residues 27-309 with and L296E mutation) under
control of the CMV promoter with a tPA signal sequence was also designed. A spike construct, encoding
a stabilized ectodomain with two Proline mutations (Spike-2P) optimized for mammalian cell expression
was obtained from the VRC, NIH (5). Genes for the heavy and light chain of the CR3022 antibody were
obtained from Genscript (USA) and cloned into the pcDNA3.4 vector
Purification of recombinant proteins expressed in Expi293F cells.
Transfections were performed according to the manufacturer’s guidelines (Gibco, Thermofisher). Briefly,
one day prior to transfection, cells were passaged at a density of 2x106cells/mL. On the day of transfection,
cells were diluted to 3.0x106cells/mL. Desired plasmids (1μg plasmid per 1mL of Expi293F cells) were
complexed with ExpiFectamine293 (2.7μL of ExpiFectamine293 per 1 μg of plasmid) and transiently
transfected into Expi293F cells. Post 16hr, Enhancer 1 and Enhancer 2 were added according to the
manufacturer’s protocol. Five days post transfection, culture supernatant was collected, proteins were
affinity purified by immobilized metal affinity chromatography (IMAC) using Ni Sepharose 6 Fast flow
resin (GE Healthcare). Supernatant was two-fold diluted with 1xPBS (pH 7.4) bound to a column
equilibrated with PBS (pH7.4). A ten-column volume wash of 1xPBS (pH7.4), supplemented with 25mM
imidazole was performed. Finally, the bound protein was eluted with a gradient of 200-500mM imidazole
in PBS (pH 7.4). The eluted fractions were pooled, and dialysed thrice using a 3-5kDa (MWCO) dialysis
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membrane (40mm flat width) (Spectrum Labs) against PBS (pH 7.4). Protein concentration was
determined by absorbance (A280) using NanoDrop™2000c with the theoretical molar extinction
coefficient calculated using the ProtParam tool (ExPASy).
Purification of recombinant protein expressed in Pichia pastoris.
20µg of pRBD vector was linearized with the PmeI enzyme by incubating at 37oC overnight (NEB,
R0560). Enzyme was inactivated (65°C, 15min) prior to PCR purification of the linearized product
(Qiagen, Germany). 10µg of linearized plasmid was transformed into Pichia pastoris X-33 strain by
electroporation as per the manufacturers protocol (Thermo Fisher). Transformants were selected on
Zeocin containing YPDS plates at a Zeocin concentration of 2mg/mL (Thermo Fisher Scientific) after
incubation for 3 days at 30oC.
10 colonies from the YPDS plate were picked and screened for expression by inducing with 1% methanol,
fresh methanol was added every 24 hrs. Shake flasks (50mL) containing 8mL BMMY media (pH 6.0)
each were used for growing the cultures for up to 120 hrs maintained at 30oC, 250rpm. The expression
levels were monitored by dot blot analysis with anti-His tag antibodies. The colony showing the highest
expression level was then chosen for large scale expression.
Larger scale cultures were performed in shake flasks by maintaining the same volumetric ratio (flask:
media) as the small scale cultures. The expression levels were monitored every 24 hrs using sandwich-
ELISA.
Cultures were harvested by centrifuging at 4000g and subsequent filtering through a 0.45µm filter
(Sartorius). The supernatant was bound to pre-equilibrated Ni Sepharose 6 Fast flow resin (GE
Healthcare). The beads were washed with 1xPBS (pH 7.4), supplemented with 150mM NaCl and 20mM
imidazole. Finally, the His tagged pRBD protein was eluted in 1xPBS (pH 7.4) supplemented with 150mM
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NaCl and 300mM imidazole. The eluted fractions were checked for purity on a SDS-PAGE. Following
his, appropriate fractions were pooled and dialyzed against 1x PBS (pH7.4) to remove imidazole.
Purification of recombinant protein from E. coli
The E. coli expression construct, eInCV01R consisted of residues 331-532 of the RBD expressed under
control of the T7 promoter with an N-terminal His tag in the vector pET15b. eInCV01R was transformed
in both E. coli BL21(DE3) (Novagen) and E. coli SHuffle T7 cells (NEB C3029H). Following cell growth
in Terrific Broth and induction with 1 mM IPTG at an OD600 of 1 at either 30 or 37 C, cells were grown
for ten hours. Expression was seen in the insoluble and soluble fractions in these two strains respectively.
Following cell lysis of the SHuffle cells, protein was purified using Ni-NTA chromatography with a yield
of about 1 mg/liter. The protein was aggregation prone and failed to bind ACE2-hFc. In the case of
BL21(DE3), following cell lysis, inclusion bodies were solubilized in buffer containing 7M Guanidine
Hydrochloride and 10mM mercaptoethanol. Protein was purified using Ni-NTA chromatography under
denaturing conditions. Protein was diluted into refolding buffer containing 0.4 M L -Arginine, 100 mM
Tris–HCl (pH 8.0), 2.0 mM EDTA (pH 8.0), 5.0 mM L-glutathione reduced, 0.5 mM L-glutathione
oxidized, but precipitated. Refolding in the absence of redox buffer was also unsuccessful.
Tag removal
The His tag was removed by subjecting proteins to digestion with HRV-3C protease (Protein: HRV-3C
= 50:1) in PBS (pH 7.4) buffer and incubating at 4°C, 16 hrs. The untagged protein (containing C-terminal
sequence: LEVLFQ) was separated from the remaining tag protein and protease by immobilized metal
affinity chromatography (IMAC) using Ni Sepharose 6 Fast flow resin (GE Healthcare). The unbound
tag-free protein was collected and protein concentration was determined by absorbance (A280) using
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NanoDrop™2000c with the theoretical molar extinction coefficient calculated using the ProtParam tool
(ExPASy).
SDS-PAGE and western blot analysis:
SDS-PAGE was performed to estimate the purity of the recombinant proteins. Protein samples were
denatured by boiling with sample buffer containing SDS, with or without DTT. For western blotting,
following SDS-PAGE, proteins were electrophoretically transferred onto an Immobilon-P membrane
(Millipore). After transfer, the membrane was blocked with 3% non-fat milk. The membrane was washed
with PBST (1xPBS with 0.05% Tween-20) and incubated with antisera raised against mRBD in guinea
pig (1:100). Following this blot was washed and incubated with α-guinea pig ALP conjugated antibody
(Sigma) at 1:5000. After washing with 1xPBST, blot was developed by BCIP/NBT Liquid substrate
system (Sigma).
Size exclusion chromatography (SEC)
A Superdex-200 10/300GL analytical gel filtration column (GE healthcare) equilibrated in 1xPBS (pH
7.4) buffer was used SEC profiles were obtained using a Biorad NGC chromatography system. The Area
under the curve (AUC) was calculated using the peak integrate tool in the Evaluation platform for various
peaks from each run
nanoDSF studies
Equilibrium thermal unfolding experiments of m331RBD (- 10xHis tag), mRBD (- 10xHis tag), pRBD (-
10xHis tag) and Spike-2P were carried out using a nanoDSF (Prometheus NT.48). Two independent
measurements were carried out in duplicate with 10-44 μM of protein in the temperature range of 15-95
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°C at 40-80% LED power and initial discovery scan counts (350nm) ranging between 5000 and 10000. In
all cases, lyophilized protein was redissolved in water, prior to DSF.
SPR-binding of immobilized ACE2-hFc / CR3022 to Spike-2P and RBD derivatives as analytes
ACE2-hFc and CR3022 neutralizing antibody binding studies with the various RBD derivatives purified
from different expression platforms were carried out using the ProteOn XPR36 Protein Interaction Array
V.3.1 from Bio-Rad. Activation of the GLM sensor chip was performed by reaction with EDC and sulfo-
NHS (Sigma). Protein G (Sigma) at 10 µg/mL was coupled in the presence of 10mM sodium acetate buffer
pH 4.5 at 30 µl/min for 300 seconds in various channels. The Response Units for coupling Protein G were
monitored till ~3500-4000 RU was immobilized. Finally, the excess sulfo-NHS esters were quenched
using 1M ethanolamine. Following this, ~1000 RU of ACE2-hFc or CR3022 was immobilized on various
channels at a flow rate of 5 µg/mL for 100 seconds leaving one channel blank that acts as the reference
channel. mRBD, pRBD and Spike-2P were passed at a flow rate of 30 µL/min for 200 seconds over the
chip surface, followed by a dissociation step of 600 seconds. A lane without any immobilization was used
to monitor non-specific binding. After each kinetic assay, the chip was regenerated in 0.1 M Glycine-HCl
(pH 2.7) (in the case of the ACE2-hFc assay) and 4M MgCl2 (in case of the CR3022 binding assay). The
immobilization cycle was repeated prior to each kinetic binding assay in case of ACE2-hFc. Various
concentrations of the mRBD (- 10xHis tag) (100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM), pRBD (- 10xHis
tag) (100 nM, 50 nM, 25 nM) and Spike-2P (-8xHis tag) (146 nM, 73 nM, 36.5 nM, 18.2 nM, 9.1 nM) in
1x PBST were used for binding studies. The kinetic parameters were obtained by fitting the data to a
simple 1:1 Langmuir interaction model using Proteon Manager.
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SPR-binding of immobilized ACE2-hFc to thermal stress subjected mammalian RBD/ Spike-2P as
analytes.
Mammalian RBD/Spike-2P protein at concentration of 0.2 mg/ml in either 1X PBS or as lyophilized
protein was subjected to thermal stress by incubation at the desired temperature in a thermal cycler for
sixty or ninety minutes respectively. Following this, lyophilized protein was resuspended in water and
SPR binding assay as described above was performed to assess the binding response using 100 nM of the
thermally stressed protein.
Limited Proteolysis
An isothermal limited proteolysis assay was carried out for mRBD, pRBD and Spike-2P using TPCK-
Trypsin at 4°C and 37°C. Substrate proteins were dialyzed in autoclaved water (MQ) and reconstituted in
the digestion buffer (50 mM Tris, 1 mM CaCl2 (pH 7.5)). ~100µg of each protein was subjected to
proteolysis with 2 µg of TPCK-trypsin (TPCK Trypsin : Vaccine candidate =1:50) incubated at two
different temperatures 4 °C and 37 °C with equal volumes of sample drawn at various time points 0, 2, 5,
10, 20, 30 and 60 minutes respectively. The reaction was quenched by SDS-PAGE loading buffer and
incubation at 95 °C and analysed by SDS-PAGE.
Guinea Pig Immunizations
Groups of four, female, Hartley strain guinea pigs, (6-8 weeks old, approximately weighing 300 g) were
immunized with 20 µg of purified antigen protein diluted in 50 µl PBS, (pH 7.4), and mixed with 50 µl of
AddaVax™ adjuvant (vac-adx-10) (1:1 v/v Antigen : AddaVax™ ratio per animal/dose) (InvivoGen,
USA). Immunizations were given by intramuscular injection on Day 0 (prime) and 21 (boost). Blood was
collected and serum isolated on day -2 (pre-bleed), 14 and 35, following the prime and boost
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immunization, respectively. All animal studies were approved by the Institutional Animal Ethics
committee (IAEC) No. RR/IAEC/72-2019, Invivo/GP/084. Although there were lockdown associated
constraints on procurement of animals, group sizes of four animals are often used in comparative
immunogenicity assessments (55).
ELISA- serum binding antibody end point titers
96 well plates were coated with immunized vaccine antigen and incubated for two hours at 25 °C (4
µg/mL, in 1xPBS, 50 µL/well) under constant shaking (300 rpm) on a MixMate thermomixer (Eppendorf,
USA). ACE2-hFc protein coating was used as a control for antigen immobilization. Following four
washes with PBST (200µl/well), wells were blocked with blocking solution (100 µL, 3% skimmed milk
in 1xPBST) and incubated for one hour at 25 °C, 300 rpm. Next, antisera (60µL) starting at 1:100 dilution
with four-fold serial dilutions were added, and plates incubated for 1 hour at 25 °C, 300 rpm. Three washes
with 1xPBST were given (200 µL of 1xPBST/well). Following this, Rabbit ALP enzyme conjugated to
anti-Guinea Pig IgG secondary antibody (diluted 1:5000 in blocking buffer) (50 µL/well) was added and
incubated for 1 hour at 25 °C, 300 rpm (Sigma-Aldrich). Subsequently, four washes were given (200 µL
of 1xPBST/well). pNPP liquid substrate (50 µL/well) (pNPP, Sigma-Aldrich) was added and the plate
was incubated for 30 minutes at 37 °C, 300 rpm. Finally, the chromogenic signal was measured at 405
nm. The highest serum dilution which had a signal above the cut off value (0.02 O.D. at 405 nm) was
considered as the endpoint titer for ELISA.
ACE2-hFc competition ELISA
96 well plates were coated with vaccine antigen and incubated overnight at 25 °C (4 µg/mL in 1x PBS,
50 µl/well) under constant shaking (300 rpm) on a MixMate thermomixer (Eppendorf, USA). Ovalbumin
(4 µg/mL in 1x PBS, 50 µL/well) coating was used as negative control for mRBD immobilization. Next,
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four washes with 1xPBST were given (200 µl/well) and wells blocked with blocking solution (100 µL 3%
skimmed milk in 1xPBST) for one hour at 25 °C, 300 rpm. Next, anti-sera (60µL) starting at a dilution of
1:10 in blocking solution, were added to sera competition wells and blocking solution alone added to the
control wells. Samples were incubated for 1 hour at 25°C, 300 rpm and three washes with 1xPBST were
given (200 µL of 1xPBST/well). An additional blocking step was performed for one hour with blocking
solution (100µL) incubated at 25°C, 300rpm. Following this, an excess of ACE2-hFc was added (60 µL
at 20µg/mL) and samples incubated for one hour at 25°C, 300rpm. Three washes were given (200 µL of
PBST/well). Next, rabbit ALP enzyme conjugated to anti-Human IgG secondary antibody (diluted 1:5000
in blocking buffer) (50 µl/well) was added and samples incubated for 1 hour at 25°C, 300 rpm (Sigma-
Aldrich). Four washes were given (200 µL of PBST/well). pNPP liquid substrate (50 µL/well) was added
and the plate was incubated for 30 minutes at 37 °C, 300 rpm. Finally, the chromogenic signal was
measured at 405 nm. The percent competition was calculated using the following equation:
% competition = [Absorbance (Control)– Absorbance (Sera Dilution)] ∗ 100 /
[Absorbance (Control)] .
Where, Absorbance (Control) is the Absorbance at 405nm of ACE2-hFc protein binding to RBD in the
absence of sera, Absorbance (Sera dilution) is the absorbance from wells where the serum dilution is
incubated with ACE2-hFc protein and mRBD.
Sandwich ELISA for monitoring RBD expression
4ug/mL ACE2 in 1xPBS pH 7.4 was coated onto ELISA strips (Thermo Fisher) for 1 hr and then blocked
with 3% BSA solution (1x PBS) for 1 hr at RT. Samples were diluted in the blocking solution and
incubated in the wells for 2 hrs at RT. The wells were incubated with anti-His Antibody (1:10000 dilution)
conjugated with Horseradish peroxidase (HRP) enzyme for 1 hr at RT following which the reaction was
visualized by adding 50µL of the chromogenic substrate, TMB (Thermo Fisher). The reaction was stopped
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26
after 20 mins with 50µL of 1M HCl and the absorbance reading at 450nm was obtained from an ELISA
Plate reader. Plates were washed with 1xPBS pH 7.4 after each step.
Negative Staining sample preparation and visualization by Transmission Electron Microscope
For visualization by Transmission Electron Microscope, Spike-2P sample was prepared by conventional
negative staining method. Briefly, Carbon coated Cu grids were glow discharged for 30 seconds and 3.5
μL of sample (0.1 mg/mL) was incubated on the grid for 1 minute. The extra sample and buffer solution
was blotted out and negative staining was performed using 1% Uranyl Acetate solution for 30 seconds.
Freshly prepared grids were air-dried for 30 minutes. The negatively stained sample was visualized at
room temperature using a Tecnai T12 electron microscope equipped with a LaB6 filament operated at 120
kV using a low electron dose. Images were recorded using a side-mounted Olympus VELITA (2K 2K)
CCD camera using defocus ranging from -1.3 to -1.5 and a calibrated pixel size 2.54 Å/pixel at specimen
level.
Reference-free 2D classification using single-particle analysis
The evaluation of micrographs was done with EMAN 2.1 (56). Around 2500 particles projections were
picked manually and extracted using e2boxer.py in EMAN2.1 software. 2D particle projections were
binned by 2 using e2proc2d.py. Reference free 2D classification of different projections of particle were
performed using simple_prime2D of SIMPLE 2.1 software (57)
CPE based viral Neutralization assay
Guinea pig sera after two immunizations (prime and boost) along with pre-immune (negative control)
samples were heat inactivated prior to the virus neutralization assay by incubating at 56 °C for one hour.
SARS-CoV-2 (Isolate: USA-WA1/2020) live virus, 100TCID50 in a volume of 50 µL was premixed with
various dilutions of the serum and incubated at 37°C for one hour in a 5% CO2 incubator. Serial dilutions
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27
of the incubated premix of virus-serum was added in duplicates into a 96 well plate containing VeroE6
cells (104/well) and cultured for 48/96 hours. After completion of incubation, the plates were assessed for
virus induced cytopathic effect (CPE) and the neutralization titre was considered as the highest serum
dilution at which no CPE was observed under the microscope.
Production of Pseudotyped SARS-CoV-2 and pseudovirus neutralisation assay
The full-length synthetic construct of Spike glycoprotein of SARS-CoV-2 (GenBank: MN908947) was
synthesized from Genewiz, UK. The complete coding sequences of the spike genes of SARS–CoV-2 and
SARS-CoV (GenBank: AY278491) lacking the endoplasmic retention signal sequence were amplified
from either the synthetic construct or cDNA and cloned into pCAGGS expression vector (pCAGGS-
SARS-2-S and pCAGGS-SARS-S). Pseudotyped coronaviruses were produced as previously described.
Briefly, the plasmids pCAGGS-SARS2-S and pCAGGS-SARS-S were transiently expressed on HEK
293T cells using Polyethylenimine (PEI) (Polysciences, USA). 24 h post-transfection, the cells were
infected with VSV∆G/GFP virus, incubated for 1 hour, and cells were washed thrice with 1xPBS and
replaced with DMEM medium containing 1% FCS and antibiotics. Pseudotyped GFP expressing
coronaviruses were harvested from the cell supernatant 24 hpi and concentrated using Amicon columns
(Merck). Then the viruses were titrated in Vero E6 cells and stored at -80 °C. A Pseudovirus neutralization
assay was performed as described elsewhere with minor modifications (58, 59). Guinea pig sera obtained
after the first immunization were tested at a dilution of 1:10-1:80 for the presence of neutralizing
antibodies to SARS-CoV-2 using pseudotyped virus. Briefly, Vero E6 cells (10,000 cells/well) were
plated in a 96 well plate (Nunc, Thermo Scientific) the day before the neutralization assay. Two-fold
serially diluted sera were prepared in 96-well plates, starting at 1:10 dilution. Pseudotyped SARS-CoV2
was diluted in Dulbecco's Modified Dulbecco's Medium (DMEM) supplemented with 1% FBS and
penicillin-streptomycin. Next, 50μl pseudotyped SARS-CoV-2 was added in each well of plates, and the
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28
plates were incubated 37°C for 1h. SARS-CoV pseudotyped virus and SARS-CoV polyclonal antibodies
(that cross-react with SARS-CoV-2) were used as a positive control. Subsequently, serum-pseudovirus
mixtures were transferred to a plate containing Vero E6 cells for one hour. Then the cells were washed
twice with 1xPBS and once with medium, and cells were grown in fresh DMEM medium followed by
incubation in a 5% CO2 environment at 37 °C for 24 hours. The neutralization titer was measured by
calculating the percentage of GFP positive cells in each well.
Declaration of Competing Interest
A provisional patent application has been filed for the RBD formulations described in this manuscript.
S.K.M, S.A., R.V, S.P, R.S are inventors. G.N, R.V are founders of Mynvax and S.P, R.S, N.G, A.U, and
PR are employees of Mynvax Private Limited.
Acknowledgements
We thank Dr. Neil King for kindly providing the ACE2-hFc fusion protein and Drs. Lynda Stuart, Dr.
Harry Kleanthous of the Bill and Melinda Gates foundation for helpful discussions. We thank Dr. Barney
Graham for kindly providing the Spike-2P construct. This work was funded by a grant from the Bill and
Melinda Gates Foundation funding to RV. The following reagent NR-52281, SARS-Related Coronavirus
2, Isolate USA-WA1/2020 was deposited by the Centers for Disease Control and Prevention and obtained
through BEI Resources, NIAID, NIH: SARS-Related Coronavirus 2, Isolate USA-WA1/2020. We also
acknowledge funding for infrastructural support from the following programs of the Government of India:
DST-FIST, UGC Center for Advanced Study, MHRD-FAST, the DBT-IISc Partnership Program, and of
a JC Bose Fellowship from DST to RV. S.K.M acknowledges the support of MHRD-IISc doctoral
fellowship. M.B. acknowledges the support of CSIR doctoral fellowship. N. Sivaji is acknowledged for
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29
his enthusiastic technical support. RS, SM and SB acknowledge the intramural funding received from
THSTI under Translational Research Program grant.
Author Contributions
R.V., S.K.M., conceptualized the work, designed the studies. S.P., R.V., G.N., planned the animal studies.
R.S., P.R., A.U., performed ELISA and ACE2-hFc competition experiments. S.K.M., K.K., S.G., S.P.,
N.G., P.K. performed mRBD, Ace2 and Spike protein expression and characterization. S.G. performed
pRBD protein expression and characterization. M.S.K., performed E.coli RBD expression and
purification. S.A. expressed and characterized the mRBD 333H mutant with assistance from M.S.K. I.P.,
S.D. provided the EM data and analysis. S.M., S.B., Ram.S. provided CPE neutralizing antibody assay
data. J.J., K.T., V.S.R, performed pseudovirus neutralization assays. M.B. contributed to antibody epitope
analyses. S.K.M wrote the manuscript with contributions from each author. S.K.M, R.V., G.N. led the
studies and edited the paper along with all co-authors.
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30
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